1. Field of the Invention
The present invention relates generally to the field of controlling the activity of cells introduced in tissue, and specifically to metabolically guiding cells introduced in tissue.
2. Background of the Technology
Metabolic engineering manipulates the rate of chemical reactions (i.e., chemical kinetics) in stirred cell populations to increase or decrease the production of desired substances. Where it is critical to the pathway of a metabolite that a certain process happen in a certain cell structure (e.g., a vacuole), that cell structure is treated as an extensionless compartment. In a larger environment, such as a stirred tank (i.e., a stirred cell population environment), maximum productivity occurs with each cell in the optimum state. The cells are also assumed to be homogeneous. When the cells are not homogeneous, the metabolic engineering is analyzed in terms of homogeneous cells and the extracellular metabolites within a uniform environment, and implied spatial variation is not brought into the kinetic analysis. Metabolic engineering manipulation is performed by: 1) genetic modification; and 2) modification of the stirred cell tank concentration of entities which affect the core kinetic processes (e.g., heat, nutrients, and waste products) or the kinetics regulatory logic (e.g., signaling compounds). For numerous cells, it is possible to apply genetic modifications for desired goals and to control the behavior of the cells. For example, it is possible to control a cell so as to cause it to multiply rapidly until it has reached an optimum level, and then divert its resources to produce, rather than grow, a desired compound. Such control involves delivering a specific molecular signal to the cell, which responds (either naturally or by genetic engineering) to that signal in a specific way.
Several types of activity are of interest in metabolic engineering. These include, but are not limited to: production (e.g., of genes, protein), secretion (i.e., production by a cell of a substance and its introduction into the body), proliferation (i.e., growth and reproduction of similar cells), and apoptosis (i.e., programmed cell death). (The term “introduction” refers to all methods and devices of delivery of any transportable entity from outside, including but not limited to: infusion, perfusion, retro perfusion, injections, and electromagnetic or ultrasound radiation. Where blood pressure or temperature is lowered by an extraction process, this is referred to as introduction of a negative quantity.) These activities must be managed over time. For example, initial rapid proliferation achieves a large population of cells, but at the optimum productive density (not necessarily the level at which proliferation is blocked by crowding or nutrient shortage), one wishes to turn their activity to production of the desired compound. All of these activities are significant also for cells introduced into a subject. For example, hypothermia (lowered temperature) can improve survival rates in implanted cells.
The metabolic engineer may wish to know and potentially control the following issues:                Whether the cells will remain close to the introduction point, how far they will spread through a target or dissipate beyond it, and whether they can migrate in sufficient quantity from a selected introduction point to a desired target point.        Whether the cells form multicellular masses that resist passive transport.        Whether the cells will attach themselves before arriving in a desired target region, preventing such arrival, or fail to attach themselves at the desired target region.        Whether and at what rate introduced cells may attack other cells (as T-cells and sometimes stem cells do naturally); how genetic engineering might be targeted to the destruction of cell types not already attacked by the subject's own cells (e.g., cancer cells or fat cells), while avoiding cells where attack is undesired or acting in a sustaining fashion for other cells (e.g., becoming oligodendrocytes replacing the myelin sheath for neurons that have lost them in such disorders as multiple sclerosis); providing trophic (nutritional) support for other cell types introduced in a procedure; or exchanging genetic material with other cells, modifying their function by in vivo genetic engineering.        Whether and at what rate the cells act to modify the extracellular matrix (ECM), laying down or removing proteins (e.g., collagen and elastin) in structures (e.g., skin and arteries), for which biomechanical properties change conspicuously with age; altering bone either in density (as in osteoporosis or its reversal) or in shape (as in the long-term response to mechanical loads).        Whether introduced stem cells or multipotent cells specialize to function as nerve cells in a region where this may be desired, or in a region where added neural activity might be disruptive.        Whether neural cells intended to replace cells lost or damaged (e.g., through stroke or degenerative disease) will form appropriate links.        Cell mobility, which can be ignored in a stirred tank.        Adhesion between cells of the type used, though this may affect aggregation, with positive or negative impact on production of the target compound.        Adhesion to other cell types or to an ECM, absent in a stirred tank, though a stabilizing porous matrix may be of use industrially.        Modification of the state or behavior of other cell types.        Modification of the ECM, by addition or subtraction of material.        Differentiation into other cell types.        Creation of synaptic connections.        
3. Related Art
Control of Cell Mobility. The system and method as discussed in U.S. Pat. No. 6,549,803, entitled “Method and Apparatus for Targeting Material Delivery to Tissue” (hereinafter referred to as “Targeting”), incorporated herein by reference, characterized a molecular population and a cell as free or bound. A free cell is not attached to the ECM. A bound cell is attached to the ECM. The present invention considers a cell as mobile. Cell mobility includes two forms: swimming and crawling. In many cases, chemically controlling swimming is performed by causing the cell to swim straight and then tumble. Tumbling is less frequent when there is an increase in the concentration of a compound to whose source the cell usually swims (e.g., a nutrient or attractant molecule). Thus, chemotaxis (i.e., the movement of cells in response to chemicals) offers a natural path that can be controlled by manipulating chemicals. Detailed modeling of metabolic control has recently been clarified, allowing more perfect control of factors, such as speed and re-engineering of the signals detected by the cell.
Swimming occurs at a characteristic velocity relative to the ambient fluid. Thus, the presence of flow due to introduction or other causes (e.g., diffusion, random walks) must be combined with flow velocity, as explained in “Targeting”. Swimming is strongly affected by the penetrability of the surrounding cells, including their anisotropy (i.e., not having properties that are the same in all directions). Thus, swimming in cross-channel directions is often more interrupted than swims along channels. Swimming can be modeled by diffusion in the presence of a tensor field of penetrability, as is described in “Targeting”. If introduced cells mutually adhere to form clumps, the cells become less free to swim, and are less able to travel passively through a porous environment of limited pore size.
In crawling, a cell stays attached to the ECM, making ambient current less relevant. Crawling involves substrate adhesion molecules, which affect movement of cells and transformations of cell states. Chemotaxis (i.e., movement of organisms in response to chemicals) may be treated by a diffusion model, though characteristic speeds are lower, and the influence of the surrounding tissue on penetrability in different directions is mediated by different physics and yields different tensors for this form of transport, which must therefore be treated separately.
Control of Cell Transport and Density. Because cells are more productive in aggregated masses, the possibility of inducing cells to form masses, rather than creating masses by manipulating environmental factors, has been discussed.
In contrast, the assumption of spatial uniformity is strikingly false. When cells are introduced into the brain, the stirred brain assumption is clearly inappropriate. Cells are typically injected or otherwise introduced via a needle or catheter. The hope is that the introduced cells will move passively, carried by the injection medium, or actively, through their own mobility—to specific regions where they are needed, and not elsewhere. In the case where the introduction device carries them to the center of the target region, the hope is that the cells will spread through a significant part of that region, and will not, in too large a proportion, migrate further. The same observation applies to the introduction of dissolved or suspended molecules, or of any other active material.
Control of Other Cell Activities. Metabolic models also impact the description and control of other cell activities, such as attack, support, or genetic modification of other cells, all of which involve sustained contact. The metabolic kinetics of differentiation (having a characteristic that is different from the original) are also under widespread study, and appear increasingly open to external control by hormones, growth factors, and other molecules that one may opt to diffuse in the course of a procedure. Unlike the industrial case, differentiation appears to be essential in many therapeutic cell introductions. The fact that cells of a maturely defined type rarely embed themselves successfully in a new environment is basic to the current emphasis on stem cells for injection. It is unlikely, however, that the target environment will always provide the stimulus for the precise transformations desired of the cells. The modification of in vitro differentiation, according to the coordinated administration of factors such as vascular endothelial growth factor (VEGF) and platelet derived growth factor subtype BB (PDGF-BB), show that the metabolic understanding needed for in vivo control can be expected as the field matures.
It is important to include heat in such a modeling system for prediction and control, not only because much metabolic kinetics is naturally modified by temperature so that cellular implant protocols already address the global temperature of the patient, but because genetic engineering permits the installation of temperature-dependent switches in kinetic pathways, governing any activity for which kinetics is sufficiently understood. Cells have been created that proliferate freely at 33° Celsius, but cease to proliferate at normal body temperature, shifting to a differentiated state. Temperature is used as a global, position-independent switch, but mild local heating is among the least invasive of measures available to the clinician. (For example, focused electromagnetic energy has been used and can result in no damage to tissues nearer the surface.)
Manipulation of the temperature field can become a powerful method of control of the activity of introduced cells that are known to respond, naturally or by arrangement, in predictable ways to differences or gradients in temperature.
In spite of the many applications of the above procedures, there has been little development of explicit numerical models for the transport and evolving density of introduced material. “Targeting” addresses the logical and implementation form of some such models and specifies particular procedures for their use in control of the delivery process.
Such developments in metabolic engineering indicate it will be increasingly practical to fine tune cell behavior over time and trigger substantial changes in cell behavior in the implantation context. The search for effective cell treatment will involve increasingly complex planning in space and time. In fact, planning in space and time has already been found useful in laboratory studies to improve implanted cell survival by including apoptosis inhibitors, antioxidants (an agent that inhibits the deterioration of materials through oxidation), and trophic factors (nutrition factors) in the introduced material. In addition, it has been found useful to bring the region below normal blood temperature during a period starting with the injection. This creates a larger search space in which to find an optimal therapy, making it unwise to rely on the best method being among those tried early. The search space will become larger again when (as we predict) the coordinated time-variation of inputs becomes necessary, and the spatial distribution of cells receives closer attention. For example, there is current debate over whether dopaminergic cells placed in the putamen suffice in treating Parkinson's, or whether they should go also to the striatum and/or the substantia nigra. The greater depth of the substantia nigra complicates the surgical procedure of direct implantation, and such multiple direct placement increases incidental damage to the brain. It is thus preferable to inject cells at a more easily and safely accessible point, in such a way that they will migrate to the actual target tissue(s). There are already instances of this approach, with cells that “know where they belong” injected with little regard for location, and migrating to the most useful place, but successes with this position-independent approach are like surface nuggets. For example, an embryonic cell ‘belongs’, strictly, at a location in an embryo. In the adult patient, acting accordingly can cause complications (e.g., in the neural connections it forms). To expect all cell types that can usefully be implanted to know where they will be therapeutically useful, and go there, is unsustainable optimism.
Thus, although it is known that neural stem cells migrate preferentially to the site of an ischemic injury (e.g., in the rat), there is a need to inject exogenous stem cells to augment the brain's own response to attempt to heal the injury, and also a need to organize and optimize this response. The present invention includes a specific approach to guiding cells and trophic/tropic factors to this end. For example, with ischemic stroke, there are compelling reasons not to transplant cells and attendant factors directly into the focal region of ischemia, due to already-present injury. As another example, one could therapeutically disrupt the blood-brain barrier (BBB), or utilize regions of the brain where the BBB is naturally modified (e.g., circum-ventricular structures) to inject materials that would otherwise be unable to penetrate brain tissue into the blood stream with a view to cure injured cells or tissue in the brain. It is important to know, and to be able to guide, the injected material to the specific locations where their therapeutic action is needed and effective.
In another example, neurotropic and neurotrophic factor pharmacology can be utilized. As spatial distribution information is available, the proper guidance of factors, as well as cells, will become critical. For example, Peptide (PACAP) is distributed within the brain parenchyma in the adult rat, while its receptors are principally located in the ventricular and sub-ventricular zones. This type of distribution and location issue is likely to be a common influence on factors needed for proper development. Thus, guiding the factors to the proper receptor locations is needed so that PACAP can function as a valuable neuroprotective agent.
If one needs to direct cells, the design of a procedure becomes more complex. Signaling compounds injected before, during, or after the cell introduction (e.g., from the same locus or other loci, using a less-invasive catheter or finer needle than required for cell delivery) may guide the cells to the desired target. It is clearly useful for the cells to be mobile when first introduced, but in the target area, the cells should attach themselves and devote resources to producing the material (e.g., dopamine) intended for that target. If a molecular agent that in a certain concentration range will trigger such a behavioral change diffuses faster (as is typical) than cells travel, it can be released from the same introduction point at an appropriate time later, to cause the majority of introduced cells to encounter a concentration of that agent in the appropriate range when they are in the appropriate region. Similarly the activity of the cells may be required for a limited time (e.g., to promote a form of healing or regrowth) or for a particular purpose (e.g., to attach themselves to a tumor and produce cytotoxic material to which cancer cells are particularly sensitive). At a certain time, such cells' work is completed, and it is preferable for them to disappear. If they do not recognize (e.g., naturally or by engineering/programming of their metabolism) the completion of their tasks and respond by apoptosis, then a molecular signal should be sent which causes them to do so. The delivery of this signal in an appropriate concentration, again, may require significant control. For example, especially in conjunction with coil design, the introduction of transcranial magnetic stimulation (TMS) allows focused delivery of electrical stimulation of particular brain regions. When appropriate cells and factors have been guided to locations, such radiative input may be used to control the rate of particular activities and pathways. The design of such multiple delivery over time and space of a plurality of cellular and molecular agents, whether fully tailored for the individual patient or as a protocol with or without some customization parameters, requires systematic model-based planning.
Image guidance permits a user injecting a substance to place a delivery instrument (e.g., a catheter) into a location (e.g., a human brain) and visualize the instrument's location relative to scan-visible structures. In present cell delivery practice, the user makes a plan for injection. This plan for injection includes the following steps: determining the composition of the cell suspension and the quantity to be loaded into the delivery system; determining the pressure or flow rate (perhaps variable) at which the substance is to be injected; and determining the time over which the flow is to be maintained. The objective of such a plan for injection is to deliver the cells in desired quantities in or to ‘target’ tissues, often while minimizing the number delivered to non-target and vulnerable tissues. Non-target tissues are tissues where the cells would be wasted, and vulnerable tissues are tissues where the cells would do harm. It is also desirable to limit excess pressure and the resulting edema (swelling) of tissue, in spatial extent, intensity, and duration.
Commonly, the plan is entered into a computer that will control the injection process, but direct hand control is also possible. The developed plan is then followed, with a change only if visually inspected images of the diffusing substance makes clear that concentration, pressure or edema is not following the course expected by the user. See, e.g., U.S. Pat. Nos. 6,026,316 and 5,964,705, which are hereby incorporated by reference. Since the evolving concentration is a three dimensional (3D) scalar field in the midst of complex 3D structures, visual inspection requires a strong grasp of the 3D relationships revealed by scan data. The prior art is limited, in that its 3D display techniques do not display these relationships clearly enough to guarantee fast comprehension and appropriately swift action by the medical user. It is rare that undesired consequences are seen fast enough to limit their scope. There is an unmet need for better 3D display techniques.
“Targeting” replaces or augments such visual inspection by enabling the computer to monitor this part of the control loop. At the simplest level of control in the invention of “Targeting”, the user chooses a position or target area and specifies an introduction plan (quantity, duration, pressure/flow-rate) to the computer. The computer then solves or determines the transport properties, on the assumption that this plan is followed, using a field of parameters P, together with available subsidiary data, such as blood pressure, to specify boundary conditions. The system displays the predicted values of concentration, pressure and edema, and the user has the opportunity to examine them at non-crisis speed, so as to determine whether they are satisfactory. If not, the user repeatedly changes the device position (actually or virtually) and/or the specified plan until an acceptable result is predicted. At this point, the plan is implemented under computer control.